1. Field of the Invention
This invention relates to cancer and reagents and methods for treating cancer. The invention generally relates to reagents and methods for inhibiting tumor cell growth, and provides said reagents and methods per se as well as in embodiments adjunct or complimentary to conventional anticancer treatments. The invention specifically provides isolated ribonucleic acid oligonucleotides, in single-stranded and double-stranded forms, that inhibit tumor cell growth, particularly in short interfering RNA (siRNA) embodiments, as well as pharmaceutical compositions thereof. Methods for using said reagents to inhibit cell growth are also provided.
2. Background of the Related Art
Tumor cell growth is known to involve expression of numerous genes, and particularly the dysregulation of that expression. Several genes having dysregulation are genes that are normally expressed during development but are improperly expressed in the tumor cell, contributing to uncontrolled growth, invasiveness and other phenotypic hallmarks of cancer.
One such gene is termed EVI1. The EVI1 (ecotropic virus integration site 1) gene, which encodes a zinc finger protein, plays important roles both in normal development and in oncogenesis. Overexpression of EVI1 has been found in certain solid tumors, such as those of the female reproductive organs, and EVI1 has been shown to be a key contributor to the emergence and clinical characteristics of myeloid malignancies, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and myelodysplastic syndromes (MDS).
Human EVI1 is localized to chromosome 3, band q26 (Morishita et al., 1990, The human EVI1 gene is located on chromosome 3q24-q28 but is not rearranged in three cases of acute nonlymphocytic leukemias containing t(3; 5)(q25; q34) translocations, Oncogene Res 5: 221-31), spans 60 kb, and contains 16 exons, with multiple alternative 5′ mRNA variants and several alternatively-spliced transcripts (Wieser, 2007, The oncogene and developmental regulator EVI1: expression, biochemical properties, and biological functions, Gene 396: 346-57). The major EVI1 form is a 1051-amino-acid protein with an apparent molecular weight of 145 kDa (Morishita et al., 1990, Unique expression of the human EVI1 gene in an endometrial carcinoma cell line: sequence of cDNAs and structure of alternatively spliced transcripts, Oncogene 5: 963-71; Matsugi et al., 1990, Identification, nuclear localization, and DNA-binding activity of the zinc finger protein encoded by the EVI1 myeloid transforming gene, Mol Cell Biol 10: 1259-64). EVI1 has multiple zinc finger domains that are organized into two sets, one each of seven and three zinc finger domains. A repression domain has been identified between the two sets of zinc finger domains, as well as an acidic region at the C-terminus (see FIG. 1). One particular transcript from the EVI1 gene, termed “the Δ324 transcript,” is an alternative splice variant of EVI1 encoding an 88-kDa protein lacking zinc fingers 6 and 7; it is found at low levels in human and mouse cells (Bordereaux et al., 1990, Alternative splicing of the EVI1 zinc finger gene generates mRNAs which differ by the number of zinc finger motifs, Oncogene 5: 925-7.). Another variant, termed “the -Rp9 variant,” lacks nine amino acids in the repression domain and is quite common in human and mouse cells.
The EVI1 protein is located in the nucleus and can bind to specific DNA sequences independently through both of its zinc finger domains (Perkins et al., 1991, EVI1, a murine zinc finger proto-oncogene, encodes a sequence-specific DNA-binding protein, Mol Cell Biol 11: 2665-74; Delwel et al., 1993, Four of the seven zinc fingers of the EVI1 myeloid-transforming gene are required for sequence-specific binding to GA(C/T)AAGA(T/C)AAGATAA, Mol Cell Biol 13: 4291-300; Morishita et al., 1995, EVI1 zinc finger protein works as a transcriptional activator via binding to a consensus sequence of GACAAGATAAGATAA(N1-28)CTCATCTTC, Oncogene 10: 1961-7). The proximal zinc finger domain recognizes a consensus sequence of 15 nucleotides consisting of GA(C/T)AAGA(T/C) AAGATAA (SEQ ID NO: 201), and EVI1 has been shown to bind directly to the Gata2 promoter through this domain (Yuasa et al., 2005, Oncogenic transcription factor Evil regulates hematopoietic stem cell proliferation through GATA-2 expression, EMBO J 24: 1976-87; Yatsula et al., 2005, Identification of binding sites of EVI1 in mammalian cells, J Biol Chem 280: 30712-22). Additionally, the binding site for this domain has a Gata1 consensus motif that may compete with Gata1 for DNA binding (Kreider et al., 1993, Loss of erythropoietin responsiveness in erythroid progenitors due to expression of the EVI1 myeloid-transforming gene, Proc Natl Acad Sci USA 90: 6454-8). Although in vitro studies showed that the distal zinc finger domain recognizes the consensus GAAGATGAG (SEQ ID NO: 202), to date, there is no report of genes that are directly regulated by EVI1 through the distal zinc finger domain.
EVI1 also interacts with several transcription regulators as shown in FIG. 2. In particular, interaction with the co-repressor CtBP is important for EVI1 function (Izutsu et al., 2001, The corepressor CtBP interacts with EVI1 to repress transforming growth factor beta signaling, Blood 97: 2815-22; Palmer et al., 2001, EVI1 transforming and repressor activities are mediated by CtBP co-repressor proteins, J Biol Chem 276: 25834-40). CtBP increases the transcriptional repression of a reporter gene by EVI1, and point mutations in EVI1 that abolish the interaction significantly decrease EVI1-mediated transcriptional repression, growth inhibition of Mv1Lu cells in response to transforming growth factor (TGF)-β, and transformation of Rat-1 fibroblasts.
EVI1 also interacts with histone deacetylases directly or through CtBP, and histone deacetylase inhibitor partially relieves transcriptional repression by EVI1 (Vinatzer et al., 2001, The leukaemia-associated transcription factors EVI1 and MDS1/EVI1 repress transcription and interact with histone deacetylase, Br J Haematol 114: 566-73; Chakraborty et al., 2001, Interaction of EVI1 with cAMP-responsive element-binding protein-binding protein (CBP) and p300/CBP-associated factor (P/CAF) results in reversible acetylation of EVI1 and in co-localization in nuclear speckles, J Biol Chem 276: 44936-43; Spensberger & Delwel, 2008, A novel interaction between the proto-oncogene Evil and histone methyltransferases, SUV39H1 and G9a, FEBS Lett 582: 2761-7). It has also been shown that EVI1 binds to the coactivators CREB binding protein (CBP) and P300/CBP-associated factor (P/CAF), and co-expression of CBP could transform a repressive effect of EVI1 on a reporter gene into a moderately-activating effect (Cattaneo & Nucifora, 2008, EVI1 recruits the histone methyltransferase SUV39H1 for transcription repression, J Cell Biochem 105: 344-52). Furthermore, it was recently shown that EVI1 associates with the histone H3 lysine 9-specific histone methyltransferases SUV39H1 and G9a (Kurokawa et al., 1998, The oncoprotein EVI1 represses TGF-β signaling by inhibiting Smad3, Nature 394: 92-6; Sood et al., 1999, MDS1/EVI1 enhances TGF-β1 signaling and strengthens its growth-inhibitory effect but the leukemia-associated fusion protein AML1/MDS1/EVI1, product of the t(3; 21), abrogates growth-inhibition in response to TGF-β1, Leukemia 13: 348-5718,19). Thus, EVI1 forms higher-order complexes with various transcriptional regulators, and these associations are important for transcriptional regulation by EVI1 (see FIG. 2).
In addition, it has been shown that EVI1 affects various signaling pathways, including the TGF-β pathway (which has been the best-characterized). TGF-β controls proliferation and cellular differentiation of most cell types and plays an important role in inhibiting tumor development. EVI1 significantly represses TGF-β-mediated activation of the p3TP-Lux reporter plasmid in HepG2 cells, and EVI1 suppresses TGF-β-mediated growth inhibition in Mv1Lu and 32D cells (Alliston et al., 2005, Repression of BMP and activin-inducible transcription by EVI1, J Biol Chem 280: 24227-37; Nitta et al., 2005, Oligomerization of EVI1 regulated by the PR domain contributes to recruitment of corepressor CtBP 2005, Oncogene 24: 6165-73). Furthermore, EVI1 interferes with the induction of endogenous genes by TGF-β and other TGF-β family members in Xenopus animal cap explants and in C2C12 cells (Alliston et al., 2005, Id.). EVI1 inhibits TGF-β signaling through at least two possible mechanisms: reduction of Smad3 activity by physical interaction, and recruitment of the co-repressor CtBP (Izutsu et al., 2001, ibid; Kurokawa et al., 1998, ibid.).
One EVI1 transcript variant, termed MDS1/EVI1, consists of sequences derived from the MDS1 gene (which is located upstream of EVI1 and is also expressed on its own) and EVI1 (Wieser, 2007, ibid.). In contrast to EVI1, MDS1-EVI1 enhances TGF-β-induced growth inhibition in 32D cells (Sood et al., 1999, ibid.) and cannot efficiently repress TGF-β-mediated activation of p3TP-Lux in HepG2 cells (Nitta et al., 2005, ibid.). The lower repressive activity correlates with a reduced ability of MDS1/EVI1, compared with EVI1, to bind to the co-repressor CtBP (Id.) (see FIG. 2).
In contrast, certain cellular proteins induce apoptosis, the disruption thereof being another way tumor cell growth is promoted. Examples of such cellular proteins include the c-Jun N-terminal kinases (JNK), which are mitogen-activated protein kinases that are responsive to various stress stimuli and play an important role in triggering apoptosis. EVI1 significantly suppresses the JNK1-mediated phosphorylation of c-Jun. Conversely, reduction of EVI1 expression using antisense oligonucleotide recovers endogenous JNK1 activity experimentally in MOLM-1 and HEC1B cells (Kurokawa et al., 2000, The EVI1 oncoprotein inhibits c-Jun N-terminal kinase and prevents stress-induced cell death, EMBO J 19: 2958-68). EVI1 physically interacts with JNK through the proximal zinc finger domain, and an EVI1 mutant lacking this domain fails to suppress JNK1 activity. EVI1 also protects cells from stress-induced cell death with dependence on the ability to inhibit JNK (Id.) (see FIG. 2).
In addition to JNK, several mechanisms have been proposed to play a role in the survival function of EVI1. EVI1 protects murine bone marrow progenitors from apoptosis by activating the Promyelocytic leukemia (Pml) gene (Buonamici et al., 2005, EVI1 abrogates interferon-α response by selectively blocking PML induction, J Biol Chem 280: 428-36). It was also reported that EVI1 suppresses TGF-β or taxol-mediated apoptosis through a phosphoinositide 3-kinase (PI3K)-Akt dependent mechanism in RIE cells (Liu et al., Evil is a survival factor which conveys resistance to both TGFβ- and taxol-mediated cell death via PI3K/AKT, Oncogene 25: 3565-75). Activator protein (AP)-1 is a transcription factor complex consisting of a Fos-Jun heterodimer or Jun-Jun homodimer. It regulates gene expression in response to a variety of stimuli, and controls a number of cellular processes including differentiation, proliferation, and apoptosis. EVI1 raises AP-1 activity and stimulates c-fos promoter activation with dependence on its distal zinc finger domain in NIH3T3 and P19 cells (Tanaka et al., 1994, EVI1 raises AP-1 activity and stimulates c-fos promoter transactivation with dependence on the second zinc finger domain. J Biol Chem 269: 24020-6). Because the distal zinc finger domain is required for EVI1-mediated transformation of Rat-1 cells, the enhanced AP-1 activity probably contributes to cell transformation by EVI1.
EVI1 is highly expressed in certain cancer cell types. The EVI1 gene is amplified in 76% of squamous cell carcinoma, the most abundant type of non-small cell carcinoma (Kang et al., 2009, Identification of novel candidate target genes, including EPHB3, MASP1 and SST at 3q26.2-q29 in squamous cell carcinoma of the lung, BMC Cancer 9: 237-52). The EVI1 gene is also amplified in lung adenocarcinoma (Id.). The expression of EVI1 was significantly higher in 19 of 25 human non-small cell lung cancer samples, as determined by real time quantitative RT-PCR, compared with non-tumor tissues (Yokoi et al., 2003, TERC identified as a probable target within the 3q26 amplicon that is detected frequently in non-small cell lung cancers, Clin Cancer Res. 9: 4705-13). A study by Brooks et al. demonstrated, by RT-PCR, that EVI1 was highly expressed in 22 of 25 human ovarian tumors samples, and 6 of 7 melanoma samples (Brooks et al., 1996, Expression of the zinc finger gene EVI1 in ovarian and other cancers, Br. J. Cancer 74: 1518-25).
Thus, because inter alia of its role in oncogenesis, EVI1 is a desirable therapeutic target for the treatment of certain cancers, and there exists a need in the art for reagents and methods for inhibiting EVI1 expression or activity or both in order to inhibit tumor cell growth, induce apoptosis in tumor cells, and otherwise provide methods for improved cancer treatment, either used alone or in conjunction with conventional anticancer agents.